Victor Stollar

Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization.

In a previous paper (G. Winkler, V. B. Randolph, G. R. Cleaves, T. E. Ryan, and V. Stollar, 1988, Virology 162, 187-196) we showed that the newly synthesized dengue-2 virus nonstructural protein, NS1, exists briefly as a monomer and then undergoes dimerization. We demonstrate here that the dimerization of NS1 is associated with a change in hydrophobicity and sedimentability of this protein. Newly synthesized monomeric NS1 is a hydrophilic and water-soluble protein which cannot be pelleted at 75,000 g. After dimerization, however, NS1 showed increased hydrophobicity in a Triton X-114 phase partitioning system and was completely pelletable at 75,000 g; these findings are consistent with NS1 becoming membrane-associated. In experiments in which infected cells were treated with tunicamycin it was shown that the glycosylation of NS1 was not required for either the dimerization or the membrane association.

Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer

Evidence is presented which indicates that the dengue-2 virus nonstructural protein NS1 (soluble complement fixing antigen) exists in infected BHK and mosquito cell cultures as part of a stable oligomer. Identification of the dissociation products of the isolated oligomer and comparison of the number of N-linked glycans in native and denatured NS1 is consistent with the idea that the high-molecular-weight form of NS1 is a homodimer. By analyzing lysates of BHK cells infected with St. Louis encephalitis virus or Powassan virus and proteins from dengue-2 virus-infected mouse brain we have demonstrated that the appearance of the high-molecular-weight form of NS1 is a general feature of flavivirus infection. It is formed between 20 and 40 min after NS1 is synthesized and before the protein passes the Golgi apparatus. Both soluble and pelletable extracellular NS1 are also found as the high-molecular-weight form.

An agent in the Aedes aegypti cell line (Peleg) which causes fusion of Aedes albopictus cells

Cell cultures of the Aedes aegypti (Peleg) cell line contain an agent that causes marked syncytium formation of Aedes albopictus cells. A plaque assay was used to measure the growth of the cell fusing agent (CFA) in A. albopictus cultures, and it was found to have a latent period of about 12 hr. It failed to replicate in or cause fusion of BHK, KB or Vero cells. The CFA was sensitive to ether and to deoxycholate and sedimented in a sucrose velocity gradient more slowly than did Sindbis virus. The preliminary evidence suggests that the CFA may be similar to the group B togaviruses.

Genetic and phenotypic characterization of the newly described insect flavivirus, Kamiti River virus

Summary. We have described in the accompanying paper by Sang, et al., ([57], Arch Virol 2003, in press) the isolation and identification of a new flavivirus, Kamiti River virus (KRV), from Ae. macintoshi mosquitoes that were collected as larvae and pupae from flooded dambos in Central Province, Kenya. Among known flaviviruses, KRV was shown to be most similar to, but genetically and phenotypically distinct from, Cell fusing agent virus (CFAV). KRV was provisionally identified as an insect-only flavivirus that fails to replicate in vertebrate cells or in mice. We report here the further characterization of KRV. Growth in cell culture was compared to that of CFAV; although growth kinetics were similar, KRV did not cause the cell fusion that is characteristic of CFAV infection. The KRV genome was found to be 11,375 nucleotides in length, containing a single open reading frame encoding 10 viral proteins. Likely polyprotein cleavage sites were identified, which were most similar to those of CFAV and were comparable to those of other flaviviruses. Sequence identity with other flaviviruses was low; maximum identity was with CFAV. Possible terminal secondary structures for the 5′ and 3′ non-coding regions (NCR) were similar to those predicted for other flaviviruses. Whereas CFAV was isolated from insect cells in the laboratory, the isolation of KRV demonstrates the presence of an insect-only flavivirus in nature and raises questions regarding potential interactions between this virus and other mosquito-borne viruses in competent vector populations. Additionally, this virus will be an important tool in future studies to determine markers associated with flavivirus host specificity.

Acidotropic amines inhibit proteolytic processing of flavivirus prM protein

Treatment of flavivirus-infected mammalian and mosquito cells with acidotropic amines (such as chloroquine, ammonium chloride, or methylamine) inhibited the normal proteolytic processing of the virus prM protein to M. As a result, virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prM protein rather than M protein. Identification of the prM protein was based on molecular weight, glycosylation, and reactivity with an anti-prM monoclonal antibody. Infected cells which had not been treated with acidotropic amines did release, along with virions which contained the mature M protein, variable amounts of virus containing the prM precursor. The relative amounts of these two types of virions were influenced both by the virus and the host cell type. Virions containing the prM protein had a lower specific infectivity than virions containing the M protein; however, in experiments with a macrophage cell line this low specific infectivity was significantly increased if the anti-prM monoclonal antibody was used to facilitate virus entry via Fc receptors. Our findings indicate that the proteolytic cleavage of prM requires an acidic environment and is necessary to generate fully infectious virus. We suggest that the cleavage of prM occurs in the acidic post-Golgi vesicles.

Association of the sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1

SVLM21 is a mutant of Sindbis virus, which in contrast to SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. We have obtained evidence that the basis of this low methionine-resistance (LMR) phenotype is the generation of an altered RNA methyltransferase with an increased affinity for S-adenosylmethionine (ado met). We now report that following the substitution of the nucleotide sequence, 126-504, from SVLM21 cDNA for the corresponding sequence of the Toto 1101 plasmid (infectious Sindbis viral RNA can be transcribed from this plasmid) we were able to generate recombinant Sindbis virus (SVMS-65a) with the LMR phenotype. (SVTOTO virus derived from Toto 1101, like SVSTD, lacks the LMR phenotype.) As was the case with SVLM21, SVMS-65a not only possessed the LMR phenotype but also showed an increased sensitivity to Neplanocin A, a potent inhibitor of S-adenosylhomocysteine (ado hcy) hydrolase. Sequencing of the nucleotide 126-504 region from SVLM21 revealed two mutations; these mutations occurred in adjacent codons and lead to two predicted amino acid changes in the SV nsPl protein; at residue 87, from Arg to Leu, and at residue 88 from Ser to Cys. Since the nucleotide sequence 126-504 lies entirely within the gene for nsP1, we conclude that the RNA methyltransferase activity generated by SV is associated with nsP1. We suggest that residues 87 and 88 in nsP1, where the amino acid changes in SVLM21 nsP1 have occurred, are at or near the binding site for ado met; we also suggest that these changes in nsP1 are responsible for the increased affinity of the SVLM21 RNA methyltransferase for ado met and thereby for the LMR phenotype. Alternatively, it is possible that the binding site for ado met is elsewhere on nsP1 or even on another protein, and that the changes at residues 87 and 88 lead to an alteration of the binding site.

Sialic acid contents of sindbis virus from vertebrate and mosquito cells. Equivalence of biological and immunological viral properties.

Abstract Sindbis virus grown in Aedes albopictus cells, in contrast to virus grown in vertebrate cells, lacks sialic acid. The presence or absence of sialic acid has no effect on the antigenic properties of these viruses as tested by complement fixation, hemagglutination inhibition, neutralization, and immune virolysis. The particle/hemagglutination unit and particle/plaque-forming unit ratios are not significantly different for virus grown in CEF (chick embryo fibroblast), BHK, or A. albopictus cells.

Sindbis virus-induced cytopathic effect in clones of Aedes albopictus (Singh) cells

Clones of Aedes albopictus cells have been isolated which, in contrast to the uncloned parental cell populations from which they were derived, show extensive cytopathic effect (CPE) after infection with Sindbis virus (SV). One such clone, Aedes albopictus LT C-7, was also shown to manifest CPE upon infection with eastern equine encephalitis virus and vesicular stomatitis virus. Yields of Sindbis virus from clone LT C-7 and from clone AIS C-3, a CPE-resistant clone, were not singificantly different. Viral RNA synthesis however, was consistently greater in the LT C-7 cells than in the AIS C-3 cells. Furthermore, in the LT C-7 cells both virus-induced CPE and viral RNA synthesis were enhanced at 34° relative to 28°; in the AIS C-3 cells there was no CPE at 28 or 34° and viral RNA synthesis was not enhanced at the higher temperature. Viral infection led to a mild inhibition of host RNA synthesis in the AIS C-3 cells but to a marked inhibition in the LT C-7 cells.

Synthesis and transmethylation of DNA in polyoma-infected cultures☆

Abstract The incorporation of label from l -methionine (methyl-H 3 ), sodium formate-C 14 , and thymidine-H 3 , into the DNA of mouse kidney cultures, labeled from the 3rd to the 36th hour after polyoma virus infection, was increased severalfold over that in the uninfected control cultures. Evidence obtained by means of several methods showed that this DNA synthesis involved cellular in addition to viral DNA. On the basis of the incorporation of the labeled methyl group from methionine into 5-methylcytosine in the DNA from both the infected and uninfected cultures, it was concluded that DNA synthesis in these cultures was accompanied by direct transmethylation. The amount of transmethylation was proportional to the amount of DNA synthesized. There was no evidence that the virus induces hypermethylation of cellular DNA. The experiments on incorporation with methionine (methyl-H 3 ) also provided evidence that the DNA extracted from polyoma virus contains 5-methylcytosine. It was calculated that the virus DNA contains approximately one-tenth the amount of 5-methylcytosine normally present in mammalian cell DNA.

The complete nucleotide sequence of cell fusing agent (CFA): Homology between the nonstructural proteins encoded by CFA and the nonstructural proteins encoded by arthropod-borne flaviviruses☆

Cell fusing agent (CFA) is an RNA virus originally isolated from a line of Aedes aegypti mosquito cells. Although our characterization of the virus many years ago showed that it resembled the flaviviruses, there was no detectable serological cross-reaction with members of the genus flavivirus. Furthermore, unlike the well-studied members of the genus flavivirus, CFA did not replicate in any of several vertebrate cell lines tested. We have now determined the nucleotide sequence of the CFA genome. Comparison of the predicted amino acid sequence of the CFA polyprotein with viral protein sequences in Genbank, has made it apparent that CFA should now be assigned to the family Flaviviridae, genus flavivirus. The homology between CFA proteins and those of other flaviviruses was highest for NS5 (45%) and NS3 (34%). Little homology was found for the structural proteins. Thus, CFA is only distantly related to the other flaviviruses for which there is sequence information; nevertheless, with respect to their hydrophobicity plots, the CFA polyprotein and the polyproteins of other flaviviruses are remarkably similar. We suggest that CFA is an insect virus, which was present in the embryos from which the Ae. aegypti cell line was established. Thus, CFA seems to be the first member of the family Flaviviridae, genus flavivirus, to be identified as an insect virus.